Hydrogen peroxide as a potential biomarker of oxidative stress is there a reliable assay

But hydrogen peroxide H2O2 can be easily detected in freshly-voided human urine, without the need for costly set-ups, and it has been proposed as a biomarker of oxidative stress.. In the

HYDROGEN PEROXIDE AS A POTENTIAL BIOMARKER

OF OXIDATIVE STRESS:

IS THERE A RELIABLE ASSAY?

MOHAMED SAH REDHA BIN HAMZAH

B.Sc.(Hons.) in Chemistry

A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE

2007

ACKNOWLEDGEMENTS

I would like to convey my deepest and most sincere appreciation to the following people from the NUS Department of Biochemistry:

Professor Barry Halliwell for his great patience and useful guidance throughout my

project despite his hectic schedule; and most importantly, for providing me with the golden opportunity to be part of his research team;

Ms Long Lee Hua for providing me with the necessary resources;

Dr Tang Soon Yew for his valuable opinions and generous sharing of knowledge;

Assist Prof Andrew Jenner for being approachable for advice;

Dr Jan Gruber, Sherry Huang, Wang Huansong, Mary Ng Pei Ern, Chu Siew Hwa, Siau Jia Ling and Li Lingzhi, for their precious contributions to the project; and

Prof Sit Kim Ping and Dr Jetty Lee for their cheerful smiles

I would like to present this work to my parents, and thank them for their love and

encouragement To Andrew Tan Kong Hui, thanks for your support too!

Through this journey with the Oxidants and Antioxidants Group, I have cultivated the habit of including fruits and vegetables in my previously unbalanced diet And I have found out that 100% atmospheric oxygen is not going to help me be a better athlete

CHAPTER 2 EXPERIMENTAL PROCEDURES

2.1 Materials

2.1.1 Reagents and instrumentation 19

2.2 Methods

2.2.6 Ferrous ion oxidation- xylenol orange version 2 (FOX-2) assay 25

2.2.8 FeTMPyP-catalysed indamine dye formation assay (FeTMPyP assay) 26

2.2.13 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay [ABTS

assay]

30

2.2.14 Preformation of ABTS+• and the quenching effect of urine 30

2.2.18 Recovery study for 2’,7’-dichlorodihydrofluorescein (DCFH) assay 34

2.2.19 Monitoring the progress of DCFH assay and the effect of catalase and

SOD

34

2.2.22 Basal urinary hydrogen peroxide measurements in human subjects 36

CHAPTER 3 RESULTS

3.1 Catalase-Based Electrochemical Method

3.2 Non-Enzymatic Chemical-Based Methods

3.2.1 Ferrous ion oxidation- xylenol orange version 2 (FOX-2) assay 46 3.2.2 FeTMPyP-catalysed indamine dye formation assay (FeTMPyP assay) 57

3.3.3 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay [ABTS

3.5 Effect Of Coffee On Basal Urinary Hydrogen Peroxide 112

ABSTRACT

Oxidative stress causes damage to the critical biomolecules in humans When left unchecked, it contributes to the development of several diseases such as cancer, diabetes, cardiovascular diseases, neurodegenerative disorders and even to the process of ageing itself Considerable debate over identifying the best biomarkers of oxidative stress is still ongoing Good biomarkers like F2-isoprostanes have been proposed to be among the most reliable but they require the use of expensive instrumentation and extensive preparation steps But hydrogen peroxide (H2O2) can be easily detected in freshly-voided human urine, without the need for costly set-ups, and it has been proposed as a biomarker of oxidative stress Obtaining urine also does not require an invasive sampling procedure In order to investigate how well H2O2 fits into the criteria of an ideal biomarker, an assay that is highly specific, sensitive and reproducible for urinary H2O2 measurement is first required In the present study, current methods of H2O2 measurement in urine samples (by FOX-2 and oxygen electrode assays) were examined, and various other peroxidase-based and non-enzymatic, chemical-based methods were developed and tested for their suitability to measure H2O2 in urine The classical oxygen electrode assay and the newly-developed, DCFH peroxidase-based assay emerged to be the two most reliable methods The DCFH assay was able to detect a basal level of H2O2 excreted by healthy individuals, with less intra-individual variation throughout the day and between different days than with the oxygen electrode assay In future, urinary H2O2 can be further studied with the DCFH assay, alongside other classical biomarkers of oxidative stress, in known

pathological conditions and to see the effect of intervention of these conditions with

antioxidant therapy Hence, the importance of a good analytical technique can never be overemphasized; in the study of biomarkers of oxidative stress, any data would be

meaningless if the method that generates them is not suitable for that application

LIST OF TABLES

Page

1.2 Biomarkers of oxidative stress/damage associated with some human

diseases

14

1.3 Data of urinary hydrogen peroxide analyzed by 3 different ways 16

3.1 Accuracy of determination of PBS solutions of H2O2 by the O2 electrode

assay

41

3.3: First study of ascorbate effect on O2 electrode assay 43

3.4: Second study of ascorbate effect on O2 electrode assay 45

3.6 Comparison of FOX-2 assay with O2 electrode assay in one individual 50

3.7 Comparison of FOX-2 Assay with O2 electrode assay in a few individuals 51

3.8 Effect of dilution of urine sample on FOX-2 assay 53

3.9 Comparison of 10xD-FOX-2 assay with O2 electrode assay 54

3.12 Effect of dilution of urine sample on HVA assay 71

3.13 HVA assay: fluorescence in different mixtures 72

3.14 Effect of dilution of urine sample on HPAA assay 74

3.16 Quenching effect of urine on preformed ABTS+• 80

3.17 Effect of dilution of urine sample on amplex red assay 85

3.18 Amplex red assay recovery study 87

3.19 Comparison of the amplex red assay with O2 electrode assay in a few

individuals

88

3.20 Effect of dilution of urine sample on DCFH assay 94

3.22 Comparison of the DCFH assay with O2 electrode assay in a few

individuals

99

3.23 Coefficient of variation of various urine samples analyzed by DCFH assay 100

3.24 Effect of dilution of urine sample on DHR assay and comparison with

DCFH assay

106

3.25 DHR assay recovery study and comparison with DCFH assay 107

3.26 Variations in H2O2 level throughout the day as measured by two assays

(DCFH and O2 electrode)

111

3.27 Effect of coffee consumption on urinary H2O2 concentration 113

LIST OF FIGURES

Page

1.2 Pathways of ROS formation, the lipid peroxidation process and the role of

glutathione and other antioxidants – Vitamin E, Vitamin C, lipoic acid – in the

management of oxidative stress

4

1.4 Chemical structure of (a) hydroxy-2’-deoxyguanosine (8OHdG) and (b)

8-iso-Prostaglandin F2α

12

3.2 A standard calibration plot for the O2electrode assay 40

3.3 A standard calibration plot for the FOX-2 assay 47

3.6 Absorbance progress of the FeTMPyP-catalyzed indamine dye formation

reaction

58

3.7 A standard calibration plot for the FeTMPyP assay 60

3.9 Pentafluorobenzenesulfonyl fluorescein (PFBSF) 65

3.10 Oxidation of HVA in the presence of HRP to a fluorescence dimer 69

3.11 A standard calibration plot for the HVA assay 70

3.12 A standard calibration plot for the HPAA assay 74

3.14 A standard calibration plot for the ABTS assay 78

3.15 Chemical structures of polyphenols and their metabolites detected in urine 81

3.16 Mechanism of action for (a) ABTS/HRP/ H2O2 and (b) ascorbic acid (AA)

with ABTS+•

82

3.17 A standard calibration plot for amplex red assay 84

3.21 Fluorescence intensity progress of the DCFH/HRP reaction with 0 to 10

3.23 Oxidation of dihydrorhodamine 123 (DHR) to rhodamine 123 103

4.1 Mechanism of DCFH-DA de-esterification to DCFH and further oxidation

to highly-fluorescent DCF by ROS and RNS

121

4.2 Schematic representation of DCFH oxidation by HRP initiated by H2O2 123

4.4 Comparison of structures of HFLUOR (dihydrofluorescein) and DCFH

(2’,7’-dichlorodihydrofluorescein), as well as their oxidized products

130

4.5 Molecular structures of ascorbic acid quenchers (AAQs) 130

LIST OF ABBREVIATIONS AND KEYWORDS

DCFH-DA 2’,7’-Dichlorodihydrofluorescein diacetate

FeTMPyP meso-Tetrakis(1-methyl-4-pyridyl)porphinatoiron(III)

FOX-2 Ferrous ion oxidation – xylenol orange version 2 (assay)

10xD-FOX-2 FOX-2 (assay) conducted on sample(s) diluted by a factor of 10

GSSG Glutathione (oxidized form)

NAD(P)+ Nicotinamide adenine dinucleotide (phosphate)

SOD Superoxide dismutase

CHAPTER 1 INTRODUCTION

1.1 FREE RADICALS AND REACTIVE SPECIES

What are free radicals? A free radical is defined as any chemical species capable

of independent existence (hence, termed ‘free’) that contains one or more unpaired

electrons in atomic or molecular orbitals (Halliwell & Gutteridge, 1999) Free radicals

and other reactive species are continuously generated in vivo during physiological and pathological processes Table 1.1 lists some of the reactive species that can be found in

vivo

Table 1.1 Nomenclature of reactive species found in vivo (adapted from Halliwell et al.,

2004b)

REACTIVE SPECIES

Reactive oxygen species (ROS)

Superoxide, O 2•- Hydrogen peroxide, H 2 O 2

Hydroperoxyl, HO 2• Hypochlorous acid, HOCl

Ozone, O 3

Peroxyl, RO 2• Singlet oxygen 1∆ g O 2

Carbonate, CO 3•- Peroxynitrite, ONOO-

Carbon dioxide, CO 2•- Peroxynitrous acid, ONOOH

Reactive nitrogen species (RNS)

Nitric oxide, NO• Nitrous acid, HNO 2

Nitrogen dioxide, NO 2• Nitrosyl cation, NO+

Nitroxyl anion, NO- Dinitrogen tetroxide, N 2 O 4

Dinitrogen trioxide, N 2 O 3

Peroxynitrite, ONOO- Peroxynitrous acid, ONOOH Nitronium (nitryl) cation, NO 2+

Alkyl peroxynitrites, ROONO Nitryl (nitronium) chloride, NO 2 Cl

1.2 REACTIVE OXYGEN SPECIES: FORMATION

By the given definition of ‘free radical’, molecular oxygen (or dioxygen) in the ground state has an electronic configuration that qualifies it to be a biradical; it has two unpaired electrons with parallel spins, each located in a different π* antibonding orbital (Fig 1.1)

Fig 1.1 Molecular orbital diagram of dioxygen (obtained from www.steve.gb.com)

The presence of unpaired electron(s) in a free radical usually confers it a

considerable degree of reactivity and this probably accounts for the reactivity of dioxygen

with other radical molecules (Valko et al., 2004) Radicals derived from oxygen represent

the most important class of radical species generated in living systems These containing radicals, together with some other non-radical, oxygen-containing

oxygen-molecules/ions, are generally termed as reactive oxygen species (ROS), which together with the reactive nitrogen species (RNS), are products of normal cellular metabolism (Table 1.1) These species are well-recognized for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems

(Valko et al., 2007)

The addition of one electron to dioxygen forms the superoxide anion radical

(O2•-) (Miller et al., 1990) Its production occurs mostly within the mitochondria due to

the ‘leakage’ of a small number of electrons from the electron transport chain which is the main source of ATP in most mammalian cells O2•- is produced from Complexes I and III located at the inner mitochondrial membrane and released into the matrix as well as

the intermembranous space (Camello-Almaraz et al., 2006) O2•- is also produced from the direct reaction of autooxidizable molecules with dioxygen, as well as through the action of certain enzymes such as xanthine oxidase and galactose oxidase (Halliwell & Gutteridge, 1999) O2•- cannot directly attack DNA, proteins or lipids, but at elevated levels, can mobilize small amounts of iron from the iron-storage protein ferritin (Bolann

et al., 1990) It can also attack the active sites of some enzymes containing iron-sulphur

clusters, causing their inactivation accompanied by iron release (Liochev, 1996)

Hydrogen peroxide (H2O2) is produced through the spontaneous or enzymatic dismutation of O2•- (2 O2•- + 2 H+ → H2O2 + O2) H2O2 can also be produced directly by several enzymes such as xanthine oxidase It is poorly reactive with most biomolecules and appears unable to directly oxidize DNA, lipids and proteins, except for a few proteins which have hyper-reactive thiol groups or methionine residues (Halliwell & Gutteridge, 1999) The danger of H2O2 largely comes from its ready conversion to the

Fig 1.2 Pathways of ROS formation, the lipid (LH) peroxidation process and the role of

glutathione (GSH) and other antioxidants – Vitamin E (T-OH), Vitamin C(AscH-), lipoic

acid – in the management of oxidative stress (adapted from Valko et al., 2007)

indiscriminately reactive hydroxyl radical (OH•), either by exposure to ultraviolet light (H2O2 → 2OH•) or through the Fenton reaction (Halliwell et al., 2000a)

Iron released by O2•- (or other transition metal ions) can participate in the Fenton reaction with H2O2 to generate OH• and the reaction can be perpetuated by any reducing agent (e.g ascorbic acid and O2•-) capable of recycling Fe3+ back to Fe2+ (Halliwell & Gutteridge, 1999):

H2O2 + Fe2+ → Fe3+ + OH• + OH

-Fe3+ + O2•- → Fe2+ + O2

With a high level of reactivity and very short half-life of approximately 10-9 s in

vivo, OH• reacts close to its site of formation (Valko et al., 2007) OH• can attack and damage all biomolecules: carbohydrates, lipids, proteins and DNA (Von Sonntag, 1987)

When lipids are attacked by OH•, the chain reaction of lipid peroxidation starts and lipid hydroperoxides (LOOH) accumulate These can be degraded in the presence of iron or copper ions (Halliwell & Gutteridge, 1999):

LOOH + Fe2+ → Fe3+ + LO• + OH

-LOOH + Fe3+ → Fe2+ + LOO• + H+

The resulting alkoxyl (LO•) and peroxyl (LOO•) radicals can damage membrane

proteins and also attack new lipid molecules to propagate lipid peroxidation

Fig 1.2 summarizes the various pathways of ROS formation

1.3 THE GOOD SIDE OF REACTIVE OXYGEN SPECIES

ROS are known to play a role in several aspects of intracellular signaling and

regulation (Valko et al., 2007) Most cell types have been shown to generate low

concentrations of ROS which act as secondary messengers in signal transduction

cascades when the cell receptors are stimulated by cytokines, growth factors and

hormones (Kamata et al., 1999) The most significant effect of ROS on signaling

pathways has been observed in the mitogen-activated protein kinase (MAPK) pathways

which involve the activation of nuclear transcription factors (Sun et al., 1996) These

factors control the expression of protective genes that repair damaged DNA, power the immune system, arrest the proliferation of damaged cells and induce apoptosis For example, the p53 protein guards a cell-cycle checkpoint, as inactivation of p53 favours uncontrolled cell division and is associated with more than half of all human cancers (Sun

et al., 1996) ROS have been implicated as second messengers involved in the activation

of nuclear factor NF-κB via tumour necrosis factor (TNF) and interleukin-1 (Hughes et

al., 2005) NF-κB regulates several genes involved in cell transformation, proliferation

and angiogenesis, and is involved in inflammatory responses (Valko et al., 2007) Fig

1.3 gives a diagrammatic summary of the activation of MAPK signaling pathways

ROS production by activated neutrophils and macrophages is a vital component

of host organism defense; the phagocytic isoform of NADPH oxidase produces O2•- and other ROS that play essential roles in killing many types of bacteria and other invaders

(DeCoursey et al., 2005) The conversion of O2 to O2•- transiently increases the O2

consumption of the cell up to 100 fold, hence the misnomer ‘respiratory burst’ (because it

is unrelated to mitochondrial respiration), while the concentration of H2O2 may reach a

level of 10-100 µM in the inflammatory environment (DeCoursey et al., 2005; Valko et

al., 2007)

ROS are also involved in other roles such as cell adhesion, redox regulation of

immune responses and as a sensor for changes of oxygen concentration (Frein et al., 2005; Waypa et al., 2005; Valko et al., 2007)

Fig 1.3 ROS-induced MAPK signaling pathways (adapted from Valko et al., 2007)

1.4 ANTIOXIDANT DEFENCES

Exposure to free radicals from a variety of sources has led organisms to evolve an antioxidant defense system comprising the following (Halliwell & Gutteridge, 1999): (a) Agents (enzymes) that catalytically remove free radicals and other ‘reactive species’ Examples are superoxide dismutase (SOD), catalase, peroxidase and ‘thiol specific antioxidants’

(b) Proteins that minimize the availability of pro-oxidants such as iron ions, copper ions and heme Some examples are protein transferrins that sequester iron so that none exists ‘free’ in plasma, caeruloplasmins that bind to plasma copper, and ferritins and metallothioneins which store excess iron and copper respectively, within cells

(c) Proteins that protect biomolecules against damage (including oxidative damage) by other mechanisms, e.g heat shock proteins

(d) Low molecular mass agents that scavenge ROS and RNS Examples are glutathione,

α-tocopherol, ascorbic acid, bilirubin and uric acid

SOD helps to diminish the direct damage caused by O2•- by accelerating its

dismutation to H2O2 The most important SOD appears to be manganese-containing SOD (MnSOD), which is located in the mitochondrial matrix; transgenic mice lacking this

enzyme die soon after birth with severe mitochondrial damage in many tissues (Li et al.,

1995) Copper- and zinc-containing SOD (CuZnSOD) is mostly located in the cytosol of animal cells while extracellular SOD (EC-SOD) is found on the cell surface of many tissues (Halliwell & Gutteridge, 1999)

H2O2 can be removed by catalase, an exclusively peroxisomal enzyme in most

tissues, as well as by glutathione peroxidase (GPx) (Chance et al., 1979):

2 GSH + H2O2 → 2 H2O + GSSG

Oxidized glutathione (GSSG) is reduced back to glutathione (GSH) by

glutathione reductase (GRed) (Chance et al., 1979):

Fig 1.2 shows some reaction pathways of the earlier discussed antioxidants

1.5 OXIDATIVE STRESS: THE BAD SIDE OF REACTIVE OXYGEN SPECIES

Free radicals and reactive species operate at a low, ‘steady-state’ concentration, measurable in cells, determined by the balance between their rates of production and their rates of removal by the antioxidant defence system which was briefly discussed earlier Oxidative stress occurs when there is a serious disturbance in this pro-oxidant –

antioxidant balance in favour of the former, leading to potential damage (Sies, 1991)

Oxidative stress can result from (Halliwell et al., 2004b):

(a) Diminished levels of antioxidants, which can arise due to mutations affecting

activities of antioxidant defence enzymes such as CuZnSOD or GPx, toxins that

deplete antioxidant defences (such as the depletion of GSH by high doses of

xenobiotics), or deficiencies in dietary minerals and antioxidants

(b) Increased production of reactive species, for example through inappropriate activation

of phagocytic cells in chronic inflammatory diseases, or exposure to elevated levels

of O2 or other toxins that are either reactive species themselves (e.g NO2•) or are metabolized to generate reactive species (e.g paraquat)

A major consequence of oxidative stress is damage to nucleic acid bases, lipids and proteins, which can severely compromise cell health and viability or induce a variety

of cellular responses through generation of secondary reactive species, ultimately leading

to cell death by necrosis or apoptosis (Dalle-Donne et al., 2006) It is widely believed that

oxidative damage to biomolecules, if left unchecked, contributes to the development of several diseases such as cancer, cardiovascular diseases, diabetes, neurodegenerative disorders and even to the process of ageing itself (Halliwell & Gutteridge, 1999)

1.6 USE OF BIOMARKERS IN OXIDATIVE STRESS MEASUREMENT

The localization and effects of oxidative stress, as well as information regarding

the nature of the ROS, may be gleaned from the analysis of discrete biomarkers of

oxidative stress/damage isolated from tissues and biological fluids Biomarkers are

defined as characteristics that can be objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacologic responses to a

therapeutic intervention (Dalle-Donne et al., 2006)

Several criteria for an ideal biomarker of oxidative stress/damage can be listed Very importantly, it must first be measurable by a robust method or assay that is specific, sensitive and reproducible for the biomarker of interest, and detectable even in normal, healthy individuals Its levels shall not vary widely in the same subjects under the same conditions at different times Ideally, it shall be predictive of the later development of the disease, though no biomarker has fulfilled this criterion as necessary experiments have

not been done (Halliwell et al., 2004a) Biomarker stability is also crucial and since most

ROS are generally too reactive and/or have a half-life too short (not more than seconds)

to allow direct measurements in cells/tissues or body fluids, their more stable oxidation

target products are measured instead, for e.g lipid peroxidation products (Dalle-Donne et

al., 2006) The biomarker must be measurable with relatively small within-assay

intrasample variation compared with between-person variations Whether obtaining the biomarker requires an invasive method or not can be an important factor for

consideration, especially when critically-ill patients are involved or when frequent

sampling is required Biological samples that have been used in previous studies include blood, plasma, urine, bronchoalveolar lavage fluid, cerebrospinal fluid, synovial fluid and

tissue biopsies (Dalle-Donne et al., 2006)

An example of a common biomarker is 8-hydroxy-2’-deoxyguanosine (8OHdG; Fig 1.4a) which is frequently measured as a biomarker of oxidative damage to DNA (Kasai, 1997) Besides the availability of this assay, other factors supporting 8OHdG measurement include (a) its formation in DNA by several reactive species such as OH•and singlet oxygen, (b) its established mutagenicity in inducing GC→TA transversions, and (c) the multiple mechanisms that have evolved to remove 8OHdG from DNA, or to

prevent its incorporation into cellular DNA, which suggests that the cell ‘perceives’ 8OHdG to be a threatening lesion that has to be removed rapidly (Kasai, 1997)

However, levels of 8OHdG are not a quantitative marker of damage to DNA by all reactive species (for example, 8OHdG is only a minor product of attack by RNS), and ROS attack on guanine residues yield not only 8OHdG, but also products such as Fapy-guanine whose amount relative to that of 8OHdG depends on the redox state of the cell and the presence of transition metal ions (Halliwell, 2000b) Hence, the same amount of free radical attack on DNA can give different levels of 8OHdG Another drawback is the artifactual generation of 8OHdG during DNA isolation from tissues, hydrolysis and analysis Consideration should also be given to other DNA base damage products which are known to be mutagenic, quantitatively more important and less ready to form

artifactually than 8OHdG (Halliwell, 2000b)

Nevertheless, 8OHdG is not readily metabolized and urinary 8OHdG is not

confounded by diet (Cooke et al., 2005)

Fig 1.4 Chemical structure of (a) 8-hydroxy-2’-deoxyguanosine (8OHdG) and (b)

8-iso-Prostaglandin F2α (b) is the most thoroughly investigated F2-isoprostane

At present, measurement of the biomarker F2-isoprostanes (Fig 1.4b) is regarded

as the most reliable approach to assess free radical-mediated lipid peroxidation in vivo (Montuschi et al., 2004) They are produced from the free radical-induced peroxidation

of arachidonic acid esterified to phospholipids (Morrow et al., 1990) Available data indicate that their quantification in either plasma or urine gives a highly precise and

accurate index of oxidative stress (Morrow, 2005) They are stable in isolated samples of body fluids, like urine and exhaled breath condensates, providing a non-invasive route for

their measurements (Dalle-Donne et al., 2006) Their measured values do not exhibit diurnal variations and are not affected by lipid content in the diet (Richelle et al., 1999)

However, F2-isoprostanes have been only reliably measured using mainly mass spectrometric-based (MS-based) methods such as gas chromatography-mass

spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS)

methods, and tandem MS methods with either LC or GC (Lee et al., 2004; Liang et al.,

2003) Though F2-isoprostanes can be measured accurately down to picomolar

concentrations with these methods, the instrumentations involved are expensive;

moreover, extensive sample preparation and clean-up (e.g phospholipid extraction, alkaline hydrolysis and derivatization) are required while great care must be taken to avoid any artifactual formation during this long processing as well as during sample

storage (Dalle-Donne et al., 2006)

Considerable debate over identifying the best biomarkers of oxidative stress is still ongoing and Table 1.2 shows many other commonly-used biomarkers of oxidative stress/damage and the diseases with which they are associated

Table 1.2 Biomarkers of oxidative stress/damage associated with some human diseases

(adapted from Valko et al., 2007)

1.7 HYDROGEN PEROXIDE AS A BIOMARKER OF OXIDATIVE STRESS

As mentioned earlier, H2O2 plays an important role as an inter- and intra-cellular signaling molecule, so a basal level of H2O2 must be present In fact, levels of H2O2 at or below about 20-50 µM seem to have limited cytotoxicity to many cell types, while levels above 50 µM have been described as cytotoxic to a wide range of cultured animal, plant

and bacterial cells (Halliwell et al., 2000a)

H2O2 has been detected in human exhaled breath condensates and the amounts of exhaled H2O2 appear greater in subjects with inflammatory lung diseases (Rosias et al.,

NO 2 -Tyr, 3-nitrotyrosine

NO 2 -Tyr, 3-nitrotyrosine

humor, probably due to the oxidation of ascorbic acid which is normally present in high concentration in these fluids (Reddy, 1990) Oxidative damage to the ocular lens leading

to cataract is slowed down by the presence of antioxidant defenses like glutathione (Lou, 2003) On the other hand, H2O2 is low or almost zero in human blood plasma (Frei et al.,

1988), likely due to its reaction with heme proteins, ascorbate and protein thiol groups, or metabolism after diffusion into erythrocytes or other cells

The excretion of hydrogen peroxide in human urine was demonstrated for the first

time by Varma et al (1990) Since then, many laboratories have confirmed the presence

of significant amounts of H2O2 in freshly-voided urine (Kuge et al., 1999; Long et al., 1999b & 2000; Hiramoto et al., 2002) Thus, it was wondered if H2O2 levels in urine might be a simple biomarker of oxidative stress While a lot of good has been said of F2-isoprostanes, and urinary/plasma o,o’-dityrosine and 3-nitrotyrosine being promising

biomarkers to be worked on (Dalle-Donne et al., 2006), costly tandem MS methods

(GC-MS/MS and LC-(GC-MS/MS) are the recommended instrumentations However, H2O2 can be easily measured in urine without the need for expensive techniques like MS or electron

spin-resonance spectroscopy, and in a shorter period of time (Long et al., 1999b) Thus,

the possibility that urinary H2O2 is a biomarker of the extent of whole body oxidative

stress is a very attractive concept to test (Yuen et al., 2003), and if the results are positive,

oxidative stress assessment could be easily done by laboratories of any scale

1.8 POTENTIAL PROBLEMS IN HYDROGEN PEROXIDE MEASUREMENT

The rationale for conducting the present study arose when urine samples from one human subject were analyzed for hydrogen peroxide The subject was a healthy non-smoker who consumed a dietary supplement pill (GNC’s MegaMen) every morning Five different samples were collected from him at the stated times (Table 1.3) within a day and were immediately analyzed by three assays, namely the oxygen electrode assay, the ferrous ion oxidation- xylenol orange version 2 (FOX-2) assay and the fluorescence assay The procedure used for the first two assays could be found in Chapter 2 The fluorescence assay was attempted using the amplex red-peroxidase assay kit (A22188) from Molecular Probes, Inc., and following protocol provided by the supplier company

Table 1.3 Data of urinary hydrogen peroxide analyzed by 3 different ways

The data, as given in Table 1.3, show that for every collection, there was a

significant intra-sample variation between the 3 assays The A22188 assay gave the lowest urinary H2O2 concentration values at all times while the O2 electrode assay gave the largest values Although the FOX-2 and O2 electrode assay gave values which

One Day / Subject X Concentration of H2O2 in µM of urine samples

Sample collection times 1100 hrs 1215 hrs 1330 hrs 1445 hrs 1600 hrs

differed considerably in magnitude, a similar trend of increase and decrease in H2O2

concentrations from 1100hrs to 1600hrs was observed between the two assays

This finding led us to many questions If these established assays have been so widely used in various types of work, why are they giving very different values of the analyte in the same sample? Which of these methods is giving the correct (or wrong) value? Or, is it possible that none of the methods is giving the right value? If these assays are not valid for urinary H2O2 measurements in the first place, can they be better tailored

to meet the specific needs of the study? Are there constituents in urine (such as excreted metabolites from the pill or diet) that can affect accurate measurements of H2O2 by these assays? Is there any chance that these interfering constituents can be identified and/or removed from urine samples which are already biochemically complex to begin with? Or, better yet, are there any other more suitable assays that can be developed to measure urinary H2O2 concentration accurately and hence be able to determine if H2O2 excreted in urine can be a suitable biomarker of oxidative stress?

1.9 IMPORTANCE OF A GOOD ANALYTICAL TECHNIQUE

H2O2 is one of the most stable ROS (O2•-, OH•, and singlet oxygen have much shorter life time), offering the opportunity to carefully quantitate the production of a ROS

by biological systems (Votyakova et al., 2004) However the accuracy of such

determinations also depends on the specificity of the assay system

In the present study, I attempt to answer as many of the questions that were raised

at the end of the previous section as possible The bottom line is that a good analytical

technique is required to measure urinary H2O2 accurately before we can confidently say whether it has the potential to be an excellent biomarker of the extent of whole body oxidative stress or not

1.10 OBJECTIVES OF PRESENT STUDY

In summary, the objectives of the present study are to:

(a) analytically validate the current methods of hydrogen peroxide measurement

in human urine samples (FOX-2 and O2 electrode assays) used in our laboratory and elsewhere;

(b) develop a new assay suitable for the measurement of urinary H2O2 that is simple, accurate, sensitive, specific, reproducible and robust; and

(c) use the assay developed in (b) to investigate if urinary H2O2 can meet as many

of the requirements set out for an ideal biomarker of oxidative stress as possible

CHAPTER 2 EXPERIMENTAL PROCEDURES

2.1 MATERIALS

2.1.1 Reagents and instrumentation

All chemicals were of the highest grade available from the stated companies: Hydrogen peroxide (H2O2; 30-35%) from Kanto Chemical Co Inc., Japan; phosphate buffered saline (PBS; 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4 and 0.24 g/L KH2PO4;

pH 7.4) from the National University Medical Institute, Singapore (NUMI);

2-[4-(hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES; 99.5% by titration) from Sigma; methanol (MeOH; HPLC grade) from Fisher Scientific; hydrochloric acid (HCl; 37% fuming) from Merck; sulfuric acid (H2SO4; min 98%) from Merck; N,N-

dimethylaniline (min 99.5%, purified by re-distillation) from Aldrich; dimethyl sulfoxide (DMSO; min 99.5%, cell culture grade) from AppliChem, Germany; ethanol (min 99.7%) from BDH AnalaR; glacial acetic acid from JT Baker; sodium hydroxide (NaOH) pellets from Merck; phosphoric acid (H3PO4; 85 %) from Mallinckrodt; 10% SDS

solution from Invitrogen; 3-methyl-2-benzothiazolinone hydrazone hydrochloride

monohydrate from Fluka; meso-tetrakis(1-methyl-4-pyridyl)porphinatoiron(III)

pentachloride (FeTMPyPCl5) from Cayman Chemicals; potassium chloride (KCl) from BDH AnalaR; disodium hydrogen phosphate (Na2HPO4) from Merck; picric acid (1% solution in water) from Aldrich; boric acid from Sigma; creatinine standard (3.0 mg/dl) from Sigma; L-ascorbate, sodium salt (98%, powder) from Sigma; ferrous ammonium

sulfate from Sigma; xylenol orange from Sigma; butylated hydroxytoluene from Sigma; pentafluorobenzenesulfonyl fluorescein (PFBSF) from Calbiochem; homovanillic acid

(HVA) from Sigma; amplex red (5 mg) from Invitrogen; p-hydroxyphenylacetic acid

(HPAA) from Aldrich; 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA; 50 mg) from Axxora Platform; 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) or ABTS from Sigma; dihydrorhodamine 123 (DHR; 10 mg) from Cayman Chemicals; catalase (EC 1.11.1.6; type C40 from bovine liver; lyophilized, 16,400 U/mg protein) from

Sigma; superoxide dismutase (SOD; EC 1.15.1.1; from bovine erythrocyte; copper and zinc-containing i.e CuZnSOD; lyophilized powder, 3700 U/mg solid) from Sigma; horseradish peroxidase (HRP; EC 1.11.1.7; lyophilized powder, 1067 U/mg solid) from Fluka; and water (MilliQ ultrapure of at least 18.2 MΩ)

Instruments used include the Molecular Devices Spectra MAX Gemini EM (for fluorescence readings), Beckman DU 640B spectrophotometer (for UV/Visible

absorbance measurements) and a Hansatech oxygen electrode

2.1.2 Human subjects

Healthy men and women aged 19 to 43 years were recruited from the Department

of Biochemistry, National University of Singapore All subjects had Body Mass Index (BMI) within the range of 17 to 24 (i.e no overweight or obese subjects were recruited) and had no history of cancer, hypertension, diabetes, cardiovascular or liver diseases Subjects were recruited regardless of race and gender All subjects were non-smokers, non-vegetarians and were not regular coffee drinkers, except for two subjects who were told to abstain from coffee for at least 14 hours before an experiment Recruited subjects

were not taking any form of oral medications or nutritional supplementations during the period of study (one of the subjects who took a dietary supplement pill daily stopped consuming them for at least 48 hours before an experiment)

Subjects were briefed on the procedures and requirements of the study All

subjects gave informed consent

2.2 METHODS

2.2.1 Preparation of hydrogen peroxide standards

A stock concentrate of approximately 30% H2O2 was freshly diluted with water to about 10 mM and the concentration was accurately determined by using the molar

extinction coefficient of 43 M-1cm-1 at the 240nm absorbance wavelength (Long et al.,

1999a) From this intermediate standard, further dilutions in water or buffer (depending

on which of the below-mentioned assays was used) were carried out to obtain the

concentration or range of concentrations of H2O2 necessary for the experiment or

standard calibration of assay (usually between 0 to 100 µM)

2.2.2 Preparation of human subjects

Subjects were on self-selected diet with no special restrictions imposed, but with the following two exceptions Subjects were not allowed to drink coffee or tea for at least

14 hours before the experiment and during the time of experiment (unless coffee is part

of the experiment) Subjects were told not to overindulge in just one or a few particular types of food one day before and during the time of experiment (for example, subjects do not make fruits, vegetables, chocolates or alcoholic beverages as a quantitatively major part of their diet) Most importantly, subjects must be physically well

2.2.3 Oxygen electrode assay

This assay was largely based on the method described by Long et al (1999b) A

Hansatech oxygen electrode (Hansatech, UK) was used Processing of signals from the electrode and recording of raw data were accomplished using a PowerLab® system and Chart™ Software, both from ADInstruments, New Zealand The electrode was set up (with saturated aqueous KCl as the electrolyte) and stabilized for 30 min with 1.5 ml of deionized water at room temperature (25oC) in the reaction chamber The chamber was then emptied and filled with 1.5 ml of urine sample After a stable baseline was recorded

on the chart, 100 µl of catalase (of type and source specified in 2.1.1) solution (10,000 U/ml in PBS buffer) was introduced to the chamber through the plunger capillary hole The net deflection was recorded on the chart and the urinary H2O2 calculated The

electrode was calibrated for O2 evolution using freshly-prepared solutions of H2O2 in water (1.5 ml each) of known concentrations

2.2.4 Recovery study for oxygen electrode assay

Urine was freshly voided in 50-ml Greiner tubes from different individuals as well as the same individuals but on different days so that a total of 8 different samples was obtained Only one urine sample was studied at a time Since the concentration of each urine was different, the creatinine level was also determined where possible (refer to section 2.2.24) 1.5 ml of neat urine was introduced into the O2 electrode chamber and analyzed as described in 2.2.3 After rinsing the chamber clean, the experiment was repeated with more 1.5 ml portions of neat urine with one additional step: varying

volumes of 5 mM H2O2 in water were added into the urine as well and dispersed by the magnetic stirrer The following table showed the volume of 5 mM aqueous H2O2 added

to 1.5 ml of urine to achieve the corresponding desired concentration of spiked H2O2

Volume of 5 mM H 2 O 2 (µl) 0.0 1.5 3.0 4.5 6.0 9.0

Each recovery was then calculated based on the response to the spiked H2O2 alone and not the total urinary H2O2 + spiked H2O2, i.e

% recovery = 100(A – B)/C

where A = experimentally-determined total concentration of H2O2 in the spiked urine,

B = experimentally-determined concentration of H2O2 in the neat urine and

C = theoretical concentration of spiked H2O2 alone in the spiked urine

The neat urine was analyzed again at the end of each spiking experiment to check for any significant increase in the level of endogenous H2O2

2.2.5 Study of ascorbate effect on oxygen electrode assay

(a) Constant [ascorbate] and varying [spiked H 2 O 2 ]

Urine was freshly voided in a 50-ml Greiner tube from one individual and

transferred into 2 separate tubes, so that each tube contained 20 ml of urine 125 µ l of freshly-made 40 mM sodium L-ascorbate (Mr = 198.1) solution was added to one tube and 125 µ l of water was added to the other 1.5 ml of the 0.25 mM ascorbate-added urine was analyzed with the O2 electrode Subsequently, 1.5 ml volumes of this urine but spiked with varying volumes of 5 mM H2O2 in water were analyzed as described in 2.2.4 The procedure was repeated with the control urine (without externally-added ascorbate)

At the end, the recovery percentages of varying levels of spiked H2O2 were calculated for both the control and the 0.25 mM ascorbate-added urine

(b) Varying [ascorbate] and constant [spiked H 2 O 2 ]

Urine was freshly voided in a 50-ml Greiner tube from one individual and

transferred separately into five 6-ml tubes Different volumes of freshly-made 40 mM sodium L-ascorbate solution were added to the 5 tubes so as to achieve the following concentrations of ascorbate in urine: 0, 0.05, 0.10, 0.20 and 0.40 mM Each 6-ml tube of urine was analyzed unspiked as well as spiked with an additional 10 µM of H2O2 (3 µl of

5 mM H2O2 in water was added to 1.5 ml of sample) using the O2 electrode At the end, the recovery percentage of 10 µM spiked H2O2 for each unique concentration of

ascorbate in urine was calculated like in 2.2.4

2.2.6 Ferrous ion oxidation- xylenol orange version 2 (FOX-2) assay

The FOX-2 assay (Long et al., 1999b and 2000) is based on the oxidation of Fe2+

by H2O2 to Fe (III), which then forms a measurable complex with xylenol orange

The following two reagents were prepared Reagent 1 was 4.4 mM butylated hydroxytoluene (BHT) in methanol; reagent 2 was 1 mM xylenol orange plus 2.56 mM ferrous ammonium sulphate in 250 mM H2SO4 One volume of reagent 2 was mixed with nine volumes of reagent 1 to make the FOX-2 reagent which was stored in the dark at 0-

40C for not more than a month

Urine sample or water (90 µl) was mixed with 10 µ l of methanol and vortexed

900 µl of FOX-2 reagent was added, vortexed and incubated for 10 minutes at room temperature Solutions were then centrifuged at 15000 g for 10 min at 4oC The

absorbance at 560 nm was read against a methanol blank As controls, the above

procedures were repeated with urine samples but adding 10 µ l of catalase solution (1000 U/ml in PBS buffer) instead of methanol The FOX-2 reagent was calibrated with known concentrations of hydrogen peroxide in water

Calculation of concentration of H2O2 in sample was done as the following:-

If Absλ=560nm (90µ l water + 10µl MeOH + 900µ l FOX-2 reagent) = AWM,

Absλ=560nm (90µ l sample + 10µ l MeOH + 900µ l FOX-2 reagent) = ASM,

Absλ=560nm (90µ l water + 10µl catalase + 900µ l FOX-2 reagent) = AWC and

Absλ=560nm (90µ l sample + 10µ l catalase + 900µl FOX-2 reagent) = ASC, then

Absλ=560nm due to H2O2 in sample = (ASM - AWM) - (ASC - AWC) = AT

So, concentration of H2O2 in sample = AT / (gradient of calibration plot)